Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.
Recapitulation of Skewed X-Inactivation in Female Ornithine Transcarbamylase-Deficient Primary Human Hepatocytes in the FRG Mouse: A Novel System for Developing Epigenetic Therapies.
复制标题
FRG 小鼠中雌性鸟氨酸转氨甲酰酶缺陷的原代人肝细胞中偏向 X 失活的重述：用于开发表观遗传疗法的新系统。
DOI：
10.1089/hum.2023.011
复制
发表时间：
2023
影响因子：
4.2
通讯作者：
Cunningham SC
中科院分区：
文献类型：
--
作者：
Cunningham SC
要实现表观遗传编辑的巨大治疗潜力，需要开发临床预测模型系统，忠实地概括目标疾病病理生理学的相关方面。在女性鸟氨酸转氨甲基酶(OTC)缺乏症患者中，携带野生型OTC等位基因的X染色体倾斜失活与疾病严重程度有关。大多数受影响的女性患者可以通过医疗手段进行管理，但也有一部分需要进行肝移植。随着表观遗传编辑技术的快速发展，重新激活沉默的野生型OTC等位基因正成为一种越来越可信的治疗方法。为此，有特权地从两名受影响的雌性婴儿身上获得移植的病变肝脏，为探索分离的患者来源的肝细胞在FRG小鼠体内的植入和扩增是否可能产生用于评价表观遗传学干预的相关模型提供了机会。这两个婴儿的肝细胞被成功地用来产生鼠-人嵌合肝脏，免疫组织化学(IHC)显示原代人肝细胞群OTC阳性或阴性，这与单个肝细胞的克隆性扩增一致，在单个肝细胞中，突变的OTC等位基因或野生型OTC等位基因被灭活。OTC阳性或阴性的人类肝细胞团的计数与一个婴儿的显著倾斜和另一个婴儿的轻微或适度倾斜一致。重要的是，来自两个婴儿的完整和分离的肝脏样本的IHC和荧光激活细胞分选分析显示出定性的相似模式，证实了嵌合的鼠-人肝脏模型重现了每个婴儿的自然状态。同样重要的是，在小鼠中诱导一种可治疗的代谢表型--口腔性酸尿，这与克隆扩增的OTC阴性的原代人类肝细胞的存在有关。我们目前正在使用这一独特的模型来探索基于CRISPR-dCas9的表观遗传学靶向策略，与高效的腺相关病毒(AAV)基因传递相结合，以重新激活非激活X染色体上沉默的功能性OTC基因。
Realization of the immense therapeutic potential of epigenetic editing requires development of clinically predictive model systems that faithfully recapitulate relevant aspects of the target disease pathophysiology. In female patients with ornithine transcarbamylase (OTC) deficiency, an X-linked condition, skewed inactivation of the X chromosome carrying the wild-type OTC allele is associated with increased disease severity. The majority of affected female patients can be managed medically, but a proportion require liver transplantation. With rapid development of epigenetic editing technology, reactivation of silenced wild-type OTC alleles is becoming an increasingly plausible therapeutic approach. Toward this end, privileged access to explanted diseased livers from two affected female infants provided the opportunity to explore whether engraftment and expansion of dissociated patient-derived hepatocytes in the FRG mouse might produce a relevant model for evaluation of epigenetic interventions. Hepatocytes from both infants were successfully used to generate chimeric mouse–human livers, in which clusters of primary human hepatocytes were either OTC positive or negative by immunohistochemistry (IHC), consistent with clonal expansion from individual hepatocytes in which the mutant or wild-type OTC allele was inactivated, respectively. Enumeration of the proportion of OTC-positive or -negative human hepatocyte clusters was consistent with dramatic skewing in one infant and minimal to modest skewing in the other. Importantly, IHC and fluorescence-activated cell sorting analysis of intact and dissociated liver samples from both infants showed qualitatively similar patterns, confirming that the chimeric mouse–human liver model recapitulated the native state in each infant. Also of importance was the induction of a treatable metabolic phenotype, orotic aciduria, in mice, which correlated with the presence of clonally expanded OTC-negative primary human hepatocytes. We are currently using this unique model to explore CRISPR-dCas9-based epigenetic targeting strategies in combination with efficient adeno-associated virus (AAV) gene delivery to reactivate the silenced functional OTC gene on the inactive X chromosome.